Foxtail millet (Setaria italica) is an ancient crop originated in China, known as Ji and Su in Chinese. Foxtail millet has the advantages of drought resistance, barren resistance, rich nutrition, dual use of grain and grass and storage resistance, which has higher water use efficiency than rice, wheat, corn and other gramineous crops. It is an advantageous crop for the structural adjustment of dry farming and planting industry, and a strategic reserve crop to deal with dry climate in the future (Diao and Cheng, 2017). Due to the relatively low yield, labor-intensive cultivation and other reasons, the planting area of foxtail millet continued to decline, which was considered as an “underutilized crop” (Zhao et al., 2019). The cultivation and utilization of foxtail millet varieties with high and stable yield is the premise for the stability and improvement of foxtail millet planting area.

Leaf photosynthesis is the basis of the formation of crop yield. The contribution of leaf photosynthesis in different leaf positions to crop yield is different. Due to the particularity of its location, the top three leaves (the flag leaf, the second leaf and the third leaf) are the main photosynthetic organs of rice, wheat and other gramineous crops at the filling stage, which is very important to the formation of grain yield. The top three leaves of rice can control about 75% of the economic yield, and the photosynthetic products of the top three leaves after heading are the main source of yield formation (Zhou, 2002; Jiang et al., 2011); If the top three leaves are removed after ear filling, the grain will become lighter and the seed setting will be reduced by more than 50% (Yang and Huang, 1979, Guangxi Agricultural Sciences, (6): 12-14). During grain filling of wheat, the photosynthetic products of the top three leaves provide more than 50% of the nutrients for the grain (Yan et al., 2004); The contribution of the flag leaf to grain yield may account for 1/3 (Xu and Zhao, 1995, Acta Agronomica Sinica, 21(2): 204-209). Leaf is also the main assimilation organ of foxtail millet, especially the top three leaves synchronized with ear differentiation, which is the main place for the formation of photosynthetic products after heading. Its growth morphology is not only an important part of ideal plant type of foxtail millet, but also the goal of high and stable yield breeding and cultivation. The experiment of foxtail millet leaf cutting showed that there were great differences in the contribution of leaves at different leaf positions to grain yield. The higher the leaf position, the greater the contribution. After the upper leaves (1~4 leaves) were cut, the ear weight lost about 30% (Lyu, 1982, Heilongjiang Agricultural Sciences, (5): 34-36). The study on the relationship between the growth morphology of foxtail millet leaves and yield showed that the top three leaves (especially the flag leaf and the second leaf) with a large leaf area were conducive to the accumulation of seed nutrients, and the wide and short leaf shape and small leaf angle were more conducive to increase the yield. It was proposed that the rational allocation of top three leaf area, leaf shape and leaf angle should be paid attention to in the breeding of foxtail millet varieties with efficient photosynthesis (Yuan, 1997, Crops, (5): 34-35).

At present, there are few studies on the relationship between the top three leaf traits and yield as well as yield related traits of foxtail millet, and there are few reports on the differences of top three leaf traits between cultivars and early lines as well as local varieties. By comparing the differences of top three leaf traits between current breeding varieties and early lines as well as local varieties, we studied the relationship between top three leaf traits and main agronomic traits as well as yield, which is of great significance to cultivate excellent foxtail millet varieties with ideal plant type. Based on the field trials of the joint identification of North China summer foxtail millet and the joint identification of Henan foxtail millet, we conducted correlation analysis and comparative analysis on the top three leaf traits, other main agronomic traits and yield of early lines, local varieties and varieties cultivated in recent years and participating in the regional test. The purpose is to clarify the change law of the top three leaf traits of foxtail millet between early lines (including local varieties) and cultivars, and the change relationship between the top three leaf traits and other agronomic traits as well as yield, in order to provide a theoretical basis for the breeding and production practice of excellent foxtail millet varieties with ideal plant type.

1 Results and Analysis

1.1 Analysis on the variation diversity of top three leaves between cultivars and early lines

From the average value, the length, width and area of the top three leaves of the cultivars were larger than those of the early lines; The variation analysis of top three leaf traits showed that the variation range of the length and width of the top three leaves of the cultivars was less than that of the early lines, while the variation range of area was larger than that of the early lines; The coefficient of variation of the length, width, area of the flag leaf, the length and width of the second leaf and the width of the third leaf of the cultivars was less than that of the early lines, while the coefficient of variation of the area of the second leaf and the length and area of the third leaf was larger than that of the early lines. From the difference of traits, the difference of the length and width of the top three leaves between the cultivars and the early lines was the same; The length was the flag leaf < the second leaf < the third leaf, and the width was the flag leaf > the second leaf > the third leaf; The difference between areas was slightly different. The areas of the cultivars were the second leaf > the flag leaf > the third leaf, and the areas of the early lines were the second leaf > the third leaf > the flag leaf (Table 1).

Table 1 Analysis on the variation diversity of top three leaves in foxtail millet

1.2 Correlation analysis of top three leaf traits between the cultivars and the early lines

The correlation analysis of the nine traits of the top three leaves (Table 2) showed that the same trait of the top three leaves had an extremely significantly positive correlation, whether it was a cultivar or an early line, such as leaf length traits, the correlation between the flag leaf and the second leaf, the flag leaf and the third leaf, and the second leaf and the third leaf was 0.918, 0.883 and 0.929 respectively in the cultivar and 0.891, 0.927 and 0.933 respectively in the early lines. The correlation between different traits was different in the cultivars and the early lines. In the cultivars, there were significant or extremely significant correlations among other traits except flag leaf length and the width of the second leaf, flag leaf length and the width of the third leaf, and the length and width of the third leaf; In the early lines, the correlation between different traits was low. It should be noted that the correlation between different traits of the same leaf was also significantly different in the cultivars and the early lines. For example, the length and width of flag leaf, the length and width of the second leaf and the length and width of the third leaf of the cultivars had an extremely significantly positive correlation; The correlation coefficient between the length and width of the same leaf of the early lines was low and could not reach the significant level. The above results showed that in the selection of these early lines, people might pay more attention to a certain trait of leaves, while in the process of breeding the cultivars, people paid more attention to the overall coordination of plant leaves.

Table 2 Correlation analysis among the 9 observed traits of top three leaves in foxtail millet Note: * and ** indicate significant at the probability level of 0.05 and 0.01, respectively

1.3 Correlation analysis of top three leaf traits and main agronomic traits between cultivars and early lines

Among the cultivars, the traits of top three leaves (especially the flag leaf and the second leaf) had high correlation with heading date and plant height; While in the early lines, except for the area of the second leaf and heading date, the length of the third leaf and plant height, the correlation between other leaf traits and heading date as well as plant height was low (Table 3). In addition, in terms of correlation coefficient, there was a positive correlation between the width of the top three leaves and plant height, while there was a negative correlation in the early lines (Table 4). Among the cultivars, there was no significant correlation between the top three leaf traits and the investigated 5 spike traits (spike length, spike diameter, single spike weight, grain weight per spike and 1000-grain weight). However, in the early lines, the length of top three leaf was significantly positively correlated with spike length, and the positive correlation between the area of the flag leaf and the second leaf and single spike weight and grain weight per spike also reached a significant level (Table 3).

Table 3 Correlation analysis on top three leaves and some main agronomic traits in foxtail millet Note: * and ** indicate significant at the probability level of 0.05 and 0.01, respectively

1.4 Correlation analysis between top three leaf traits and yield of the cultivars

The correlation analysis between the yield and the top three leaf traits of the cultivars showed that except the flag leaf width, there was a significantly negative correlation between the other 8 leaf traits and the yield, among which the negative correlation between the area of the second leaf as well as the width and area of the third leaf and the yield reached a very significant level (Table 4). In order to further analyze the relationship between top three leaf traits and yield, we divided the varieties into two groups according to the size of top three leaf area: one group had large top three leaf area and the other group had small top three leaf area. After correlation analysis, it was found that the negative correlation between yield and top three leaf traits was more obvious in the group with large top three leaf area, while it was not significant in the group with small top three leaf area, and the correlation coefficient between top three leaf width, flag leaf area and yield of the latter changed to be positive (Table 4). Because many early lines fell down seriously during the filling period, which had a great impact on the yield, this study did not analyze the relationship between the top three leaf traits and yield of the early lines.

Table 4 Correlation analysis on yield and the 9 observed traits of top three leaves in foxtail millet cultivars Note: * and ** indicate significant at the probability level of 0.05 and 0.01, respectively

2 Discussion

Due to continuous directional selection, variety improvement will continuously reduce the genetic diversity of phenotype, so as to improve some traits (Tian et al., 2005). This study found that the 9 traits of the top three leaf size (leaf length, leaf width and leaf area) of the cultivars were higher than those of early lines, but the variation range and coefficient of variation were generally lower than those of early lines, which was consistent with the previous results of the reduction of the diversity index of morphological traits of the cultivars by comparing with the genetic diversity of foxtail millet germplasm (Tian, 2010; Jia et al., 2013; Wang et al., 2016; Xiang et al., 2018). Leaf size is an important indicator of crop morphogenesis. As functional leaves, the top three leaves are considered to be the main source of carbohydrates. Their appropriate size is the key to improve photosynthesis and energy accumulation. The appropriate increase of the top three leaf size of foxtail millet is conducive to photosynthesis, so as to effectively improve the yield of foxtail millet varieties. This study also found that the correlation between the 9 traits of the top three leaves in cultivars was very high, while it was generally low in early lines, which reflected that early lines may focus on the phenotypic selection of a certain trait of leaves, so as to be retained as germplasm resources, while the cultivars paid more attention to the overall coordination of plants in the process of breeding to ensure the yield, so as to be used in production. The above results showed that in addition to the increase of the size of the top three leaves, we should also improve the overall coordination of plant leaves in the breeding of foxtail millet varieties, and the appropriate size and reasonable distribution of the top three leaves were more conducive to photosynthesis and energy accumulation, which was conducive to the improvement of the yield of foxtail millet.

The correlation analysis between the top three leaf traits and the main agronomic traits found that the correlation between the top three leaf size and heading date as well as plant height traits of the cultivars was significantly higher than that of early lines, which showed that the growth of leaves and plants were more coordinated after the improvement of foxtail millet varieties. From the correlation coefficient and significance level, the correlation between the top three leaf size and the five spike traits such as spike length, spike diameter, single spike weight, grain weight per spike and 1000-grain weight of the cultivars was significantly lower than that of the early lines, suggesting that the improvement of foxtail millet varieties weakened the correlation between the top three leaf size and spike traits. The most obvious was that in the early lines, there was a significant positive correlation between the top three leaf area and the grain weight per spike, which was closely related to the grain yield, while in the cultivars, the correlation coefficient between these two traits was very small and can not reach the significant level. Early studies also showed that there was a significant positive correlation between the top three leaf area and grain weight per spike (Zheng and Wu, 2005; Li et al., 2007, Shaanxi Journal of Agricultural Sciences, (1): 9-11), which maybe because the early variety improvement often paid attention to individual yield and was easy to breed large spike and sparse planting varieties, and large leaves were more likely to produce large spike varieties. In this study, the significant positive correlation between top three leaf area and grain weight per spike of early lines was just in line with this fact. The cultivars used in this study were mainly cultivated in recent years. In the process of breeding these varieties, breeders weakened the breeding strategy of large spike and sparse planting, and paid equal attention to the traits of both population and individual, so as to meet the needs of modern agricultural production (Zhang et al., 2017). In previous experiments, the planting density was low, the traits of individuals in the field could be fully expressed under the condition of low density, and the relationship between the top three leaf size and spike traits was easier to be reflected; This experiment was carried out at the planting density of 600 000 plants/hm². The probability of mutual covering of leaves between plants increased. Too large leaves increased the degree of covering of leaves, which was not conducive to photosynthesis and material accumulation. In addition, the current breeding of foxtail millet varieties usually goes through the selection of high-density planting conditions. If the top three leaves are too small, it is not conducive to individual growth, while if the top three leaves are too large, it is not conducive to population growth. Therefore, compared with early varieties or lines, the yield of the current cultivars was effectively improved, but the variation range of the length and width of the top three leaves has been narrowed, thus, the correlation between the top three leaf size and the spike traits was reduced. The above results showed that under the actual needs of high-density variety breeding and production, foxtail millet variety improvement improved its own regulation ability, high yield and stable yield, and ensured the population yield under the condition of high-density planting by continuously weakening the correlation between the top three leaf size and spike traits of the individual.

Although the correlation between the top three leaf size and the spike traits of the current foxtail millet varieties was reduced, and the yield of the tested varieties in this study was significantly negatively correlated with the top three leaf traits. It was speculated that the population yield of the varieties with larger top three leaves would be negatively affected under the planting density of 600 000 plants/hm² in this experiment. The results of grouping analysis of varieties according to the size of top three leaf area did show that the negative correlation between yield and top three leaf traits existed only in the group with large top three leaf area, while in the other group with small top three leaf area, there was not only a lack of this negative correlation, but also the relationship between leaf width, area and yield tended to change to a positive correlation. These results showed that under the condition of specific planting density, when the top three leaf area of foxtail millet varieties exceeded a certain threshold, the population yield would be significantly inhibited. It showed that under the current requirements of high-density mechanized agricultural production, foxtail millet varieties with too large leaf area were not suitable. Appropriately reducing the leaf area, especially the top three leaf area, was more conducive to the adaptation of varieties to the requirements of agricultural production. The study on the variation law of leaves of main varieties of other gramineous crops cultivated in different years found that the upper leaves became smaller: during the replacement of main varieties of wheat in Shandong Province, the evolution of flag leaf area generally showed a downward trend (Bi et al., 2012, Shandong Agricultural Sciences, 44(6): 63-65), the length of the top three leaves of early indica rice cultivated in different years tended to become shorter (Wan et al., 2005), with the progression of years, the length of the flag leaves of rice varieties in Jilin Province became shorter and the area decreased (Zhao et al., 2011). In addition, in the process of variety replacement of gramineous crops, breeders also put forward requirements for the ideal posture of leaves. It is generally considered that the flag leaf of high-yield varieties is upright, the second leaf is a little inclined, the third leaf is inclined, and the leaf basal angle and leaf open angle are gradually increased from top to bottom, so as to increase the light capture of leaves and improve the photosynthetic utilization rate of populations (Zhang et al., 2004; Liu et al., 2010). As to foxtail millet, it was also mentioned an ideal plant type with a “tower” structure in which the top three leaves should be wide and short, and the length distribution should be short at the top and long at the bottom (Yuan, 1997; Li et al., 2007, Shaanxi Journal of Agricultural Sciences, (1): 9-11; Luo et al., 2012). Combined with the requirements of the current development of high-density mechanization for the ideal plant type of millet, and drawing lessons from the change law of leaves in the evolution of other gramineous crop varieties, the top three leaf area should not be too large in the breeding of foxtail millet varieties, and the appropriate leaf area can be ensured by reducing the leaf length and moderately increasing the leaf width. On the premise of meeting the conditions of high-density planting, the top three leaf area should be selected according to the plant height, leaf angle and other traits, so as to reduce the influence of upper leaf covering on the lighting and ventilation of lower leaves; At the same time, the single spike of the plant should not to be too large and heavy, which will help the whole plant to be in an upright or semi upright state, and then improve the yield of the population through the increase of planting density and effective photosynthetic efficiency.

3 Materials and Methods

3.1 Test materials

The test materials were 41 foxtail millet cultivars and 30 early lines (Table 5). The cultivars were mainly the varieties cultivated in recent years and participating in the regional test (No. 1~24 were the varieties participating in the regional test), and the early lines were the early lines or local varieties formed before 2000. The regional test varieties were provided by the participating units, and the rest were provided by Cereal Crops Institute, Henan Academy of Agricultural Sciences.

Table 5 The foxtail millet cultivars (lines) used in this experiment

3.2 Test design

The experiment was conducted in Henan Modern Agricultural Research and Development Base (Yuanyang) from June to October in 2018. The planting density is 600 000 plants/hm², the length of the row was 5 m, and the row spacing was 0.4 m. There were 24 varieties participating in the regional test, 8 rows in each plot, and the plot area was 16 m²; as to other varieties (lines), there were 4 rows in each plot, and the plot area was 8 m², which was randomly arranged and repeated for 3 times. The previous crop of the experimental land was wheat, the soil type was loam, and the fertility was medium. The experimental materials were sown on June 17 and harvested on September 23.

3.3 Determination of traits

The heading date and top three leaf traits were recorded in the field investigation. The measurement of top three leaf traits was carried out one week before harvest. Five representative plants were selected from each plot to measure the widest part and length of the flag leaf, the second leaf and the third leaf, which was recorded as leaf width and leaf length respectively, and leaf area = leaf length × leaf width × 0.75. At maturity, five plants with uniform growth and consistent plant type were randomly selected from each plot and brought back to the laboratory for seed test. The plant height, spike length, spike diameter, single spike weight, grain weight per spike and 1000-grain weight were measured, and the yield of the varieties participating in the regional test was measured.

3.4 Data statistics

The data were analyzed and processed by Excel 2007 and SPSS 22 software.

Authors’ contributions

DST and ZCC were the executors of the experimental design and research of this study; DST completed data analysis and wrote of the first draft of the manuscript; QN, SYH, MCY, WCY, CYX, and RZX participated in the experimental design and analysis of the experimental results; LJX was the conceiver and person in charge of the project, guiding experimental design, data analysis, manuscript writing and revision. All authors read and approved the final manuscript.

Acknowledgments

This study was jointly funded by Special Fund Project of Ministry of Agriculture/Ministry of Finance for Modern Agricultural Industrial Technology System (nycytx-CARS-06) and Independent Innovation Project of Henan Academy of Agricultural Sciences (2020zc12).

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